1. Field of the Invention
The invention relates to a high-speed process for amplifying DNA, and a companion apparatus for automating this process, based upon the Polymerase Chain Reaction (PCR). Specifically, the present invention relates to a process in which pressurized gas is used to heat and cool a biochemical reaction chamber. This novel process has been successfully automated, as demonstrated by its ability to amplify DNA from picogram to microgram quantities on an unprecedented time scale.
2. Significance
Anyone who has ever driven an automobile or flown in a jet airplane can attest to the speed and reliability of pressurized gas machines. Pressurized gas devices include internal combustion engines, jet turbines, rockets, pneumatic airtools, and gas chromatographs. However, gas machines are not widely used in biochemistry. Described herein is a novel process for high-speed Polymerase Chain Reaction amplification of DNA, which relies upon the use of a pressurized gas thermocycler with high speed valves.
Five features of this pressurized gas thermocycler are important: (1) It is the fastest automated PCR device ever built; 30 cycles of amplification of an 85 base pair (b.p.) DNA fragment were carried out in 78 seconds. (2) With proper engineering, it can be made even faster. (3) It is compatible with on-line, fluorescent dye-based DNA detection optics. (4) Unlike any device which has been previously described, the speed of DNA thermocycling is limited by the biochemistry rather than the dead time of the thermocycler. In high-speed gas phase PCR, the rate of Taq Polymerase elongation (xcx9c80 nucleotides/sec at 72xc2x0 C.) is rate-limiting. Theoretically, if faster DNA Polymerases ( greater than 1000 nucleotides/sec) can be found which are compatible with high-speed gas phase PCR, then even faster thermocycling times ( less than 10 sec/30 cycles) are possible. (5) As an added benefit, high-speed gas phase PCR is generally more accurate than slower methods, probably because false reaction products have so little time to anneal and/or elongate.
Pressurized gas thermocyclers should prove especially useful in the diagnosis of life-threatening diseases where speed is essential. The present invention has major implications for DNA-based diagnoses used in biomedical research, genetics, molecular medicine, agriculture, veterinary science, and forensics.
3. The Background Art
[a] The Polymerase Chain Reaction. In order to understand how and why pressurized gas thermocyclers were built, one must first understand the Polymerase Chain Reaction (PCR) and how it has previously been automated. The Polymerase Chain Reaction is one of the most widely used techniques in molecular biology (U.S. Pat. No. 4,683,202 to Mullis; Saiki et al., 1985; Erlich, 1989; Mullis et al., 1994). PCR-amplified DNA can be used to diagnose mutations responsible for human genetic diseases (Kogan et al., 1987), in blood and tissue typing (Saiki et al., 1989a), or to detect pathogens responsible for important infectious diseases (Persing et al., 1993).
In a typical PCR reaction, template DNA sequences lying between the ends of two defined oligonucleotide primers can be amplified in 1 to 2 hours. Three sequential steps are normally employed: (i) double-stranded DNA is denatured (D) to a single-stranded form at a high temperature (90xc2x0 C. to 95xc2x0 C.), (ii) the resulting single-stranded DNA strands are annealed (A) to oligonucleotide primers at xcx9c40xc2x0 C. to 60xc2x0 C., and (iii) primer template complexes are elongated (E) using a thermostable DNA Polymerase such as Thermus aquaticus (Taq) Polymerase at xcx9c72xc2x0 C. (Saiki, 1989b).
One cycle of these three steps (denaturation/annealing/elongation) results in a two-fold amplification of a DNA fragment whose 5xe2x80x2 and 3xe2x80x2 ends are defined by sequence-specific annealing of the oligonucleotide primers to the DNA template. Therefore, 30 PCR cycles result in a 230-fold (xcx9c106-fold) amplification of a particular DNA sequence. DNA is thus amplified from picogram to microgram amounts, which can be detected by standard analytical methods, such as gel electrophoresis, DNA hybridization, or optically.
[b] Automated PCR Instruments. A variety of machines have been built which automate the three-step PCR amplification process (Oste, 1989; Oste, 1994; Newton, 1995; Johnson, 1998). Generally, these devices may be classified into two categories: robotic devices which move the DNA samples to the heat; and thermocyclers which bring the heat to the samples.
Robotic devices such as Stratagene""s ROBOCYCLER move tubes containing PCR reaction samples to and from a series of heat baths, which are thermostated at different temperatures. Although these devices may be useful in certain research applications, they are incapable of high-speed PCR. They require greater than 60 minutes for 30 cycles of amplification.
Since the late 1980s, thermocyclers have become familiar devices in many biochemistry laboratories. Most commercially available PCR devices (Perkin-Elmer, MJ Research, Ericomp, Techne, Eppendorf, BioRad, Hybaid) are thermocyclers (Johnson, 1998). In general, two types of thermocyclers are employed: programmable heat blocks and hot-air thermocyclers.
[c] Programmable Heat Blocks. Most thermocyclers resemble xe2x80x9cwaffle irons.xe2x80x9d They are heat blocks with holes in them where plastic reaction tubes are heated and cooled under electronic control. Several such devices have been described by Johnson (1998). The problem with this type of design is that one spends most of ones"" time waiting for a block of metal to heat up or cool down. Like the waffle chefxe2x80x94who spends most of his time heating up the waffle iron beforehand, or cooling it off afterwardsxe2x80x94very little time is spent actually cooking the waffles. For example, in the MJ Research PTC-150 thermocycler (Watertown, Mass.), 14 seconds/cycle is lost in transition between D, A, and E temperatures (xcx9c94xc2x0 C., 55xc2x0, and 72xc2x0).
Many commonly employed PCR protocols spend one minute at 94xc2x0 C. (denaturation), one minute at xcx9c55xc2x0 C. (annealing), and one minute at 72xc2x0 C. (elongation). For example, in the original PCR method used by Cetus workers (Saiki et al., 1989b), a 536 b.p. xcex2-globin DNA fragment was amplified using 30 cycles of (1 min at 94xc2x0 C., 1 min at 55xc2x0 C., 1 min at 72xc2x0 C.). The active duty time for this thermocycling protocol is only xcx9c3.5 minutes=210 seconds. This is the time needed to enzymatically copy a 536 b.p. template 30 times at an elongation rate of xcx9c80 nucleotides/sec (Innis et al., 1988; Gelfand and White, 1990).
Commercially available heat block thermocyclers (Perkin-Elmer, Ericomp, MJ Research, Eppendorf, Techne, BioRad, Snark Technologies) require 20 to 25 seconds to cool from 94xc2x0 C. to 55xc2x0 C. and another 14 to 20 seconds to heat from 55xc2x0 C. to 94xc2x0 C. (Johnson, 1998). Therefore, the xe2x80x9cdead timexe2x80x9d for each PCR cycle is another 40xc2x15 seconds per cycle. As shown in FIG. 4, commonly employed thermocycling protocols require (220 seconds/cyclexc3x9730 cycles)=6600 seconds=110 minutes (Saiki et al., 1989b). Only xcx9c3.5 minutes of this xcx9c2 hours is productively focused on the PCR process.
[d] Hot-Air Thermocyclers. In order to overcome the long transitional dead times of heat blocks, hot-air thermocyclers have been constructed which allow 30 cycles of PCR amplification to be carried out in as little as xcx9c10 to 30 minutes. Wittwer and his colleagues have carried out considerable engineering groundwork to optimize rapid DNA amplification in hot-air PCR thermocyclers (Wittwer et al., 1989; Wittwer and Garling, 1991; Wittwer et al., 1994). The rate-limiting step in the three-step PCR reaction sequence (denaturation/annealing/elongation) is the rate of DNA Polymerase elongation. At an elongation rate of 80 nucleotides/sec by Taq Polymerase (Innis et al., 1988; Gelfand and White, 1990), less than one second per cycle is actually needed to amplify DNA fragments shorter than 100 b.p. using xcx9c20 mer primers. For example, only about five seconds per cycle were needed to copy a 536 b.p. xcex2-globin amplicon through 30 PCR cycles (Idaho Technology, 1995).
In commercial hot-air thermocyclers, first built by Idaho Technology (Idaho Falls, ID), the reaction time needed for one PCR cycle of denaturation/annealing/elongation was substantially reduced because: (i) the device had very low thermal mass; (ii) gaseous phase heat transfer from hot-air to the aqueous reaction was carried out in thin-walled capillary tubes; and (iii) the denaturation and annealing times during the PCR cycle were minimized. Using a PCR protocol of 30 cycles of [0 sec 94xc2x0 C. (denaturation), 0 sec 55xc2x0 C. (annealing), 5 sec 72xc2x0 C. (elongation)], a 536 b.p. xcex2-globin DNA fragment was amplified in 9.9 minutes (Idaho Technology, 1995). This rapid hot-air thermocycling protocol was therefore xcx9c220/19.8=11 times faster than the original protocol of Saiki et al. (1989b) for PCR amplification of a 536 b.p. xcex2-globin DNA fragment in a conventional heat block thermocycler. Although the design of hot-air thermocyclers of Wittwer et al. (U.S. Pat. No. 5,455,175; Wittwer et al., 1990; 1994) is admirable, it is not optimal.
First of all, Wittwer et al. (1990) have stated that xe2x80x9cAir is an ideal heat transfer medium which can change temperature quickly because of its low density.xe2x80x9d This statement is demonstrably incorrect. Heat transfer by conduction is described by the Fourier Equation:
q=xe2x88x92kA[dT/dx]
where q is the heat flux, k is the heat transfer coefficient, A is the surface area, and dT/dx is the thermal gradient. This mistake is repeated by Zurek et al. (1996; U.S. Pat. No. 5,576,218) who also rely exclusively on forced hot air as a heat transfer medium.
The basic laws governing heat transfer in the gas phase have been known since Fourier (1822) and are taught in standard mechanical engineering textbooks (Bosworth, 1952; Azbel, 1984; Chapman, 1984). For a given thermal gradient dT/dx in a thermodynamic system of fixed surface area A, the heat flux q is directly proportional to the heat transfer coefficient k; in other words, dT/dx=xe2x88x92q/kA. The negative sign indicates that heat is lost from the system. The heat transfer coefficient of helium was measured by Ubbink (1947) and is listed in tables published by Johnston and Grilly (1946), Bosworth (1952), Azbel (1984), and Chapman (1984). As shown in FIG. 1, helium has a heat transfer coefficient seven times that of air. Neon transfers heat about twice as fast as air.
Therefore, prior art hot-air thermocyclers use the wrong gas for rapid heat transfer. Among the non-combustible gases, air is available; helium is optimal. U.S. Pat. No. 5,455,175 to Wittwer et al. incorrectly assumes that the same gas (air) should be used for heating the reaction chamber, cooling the chamber, or holding its temperature at a fixed value. In fact, as shown in the temperature versus time profile in FIG. 8b, high k gases, such as helium, are superior for heating/cooling the reaction chamber, whereas low k gases, such as air or CO2, afford better thermal control when holding the temperature for several seconds during elongation at xcx9c72xc2x0 C. (see FIG. 5b and 8b). In other words, different gases are optimal for different steps of the PCR process.
Secondly, U.S. Pat. No. 5,455,175 to Wittwer et al. and U.S. Pat. No. 5,576,218 to Zurek et al. specify the use of air (but no other gas) at atmospheric pressure (but no other pressure) for gas phase PCR. Not only is air a relatively poor heat transfer gas, but it need not be used at atmospheric pressure. Gas phase PCR at atmospheric pressure is convenient; but the process is orders of magnitude faster at elevated pressure (P greater than 1 atm; see FIGS. 4, 5b and 8b).
For example, in the forced hot-air thermocycling process of Zurek et al. described in U.S. Pat. No. 5,576,218, xe2x80x9cthe sample could be heated from 50xc2x0 C. to 85xc2x0 C. within 12 to 15 seconds . . . by injecting cooling air at a substantially lower temperature than the target temperature, for example 22xc2x0 air, the sample was cooled from 85xc2x0 C. to 50xc2x0 C. in approximately 60 to 75 seconds.xe2x80x9d Therefore, the method of Zurek et al. described in U.S. Pat. No. 5,576,218 requires at least (12+60)=72 seconds per cycle just to heat and cool the gas (air), regardless of whether any useful biochemistry has taken place. In the present invention (see FIGS. 8a and 8b), 30 cycles of PCR amplification are achieved in as little as 78 secondsxe2x80x94about the same time needed for one cycle using the method of Zurek et al. described in U.S. Pat. No. 5,576,218.
Third, it has been unnecessarily assumed by Wittwer et al. (U.S. Pat. No. 5,455,175; Wittwer et al. 1990; 1994) that the heat chamber and reaction chamber are the same thing (see FIG. 2).
Altogether, for efficient heat transfer in the gas phase, hot-air thermocyclers utilize the wrong gas, wrong pressure, and wrong configuration of heat chamber to reaction chamber. Much faster gas phase thermocyclers can be designed using pressurized gas, particularly pressurized helium.
A process for amplifying DNA using pressurized gas and electronic valves has not been previously described. Both the high-speed pressurized gas amplification process and a companion apparatus for its automation are the primary objects of the present invention.
Specifically, in view of the above described state of the art, the present invention seeks to realize the following objectives.
It is an object of the present invention to provide a process for amplifying DNA so that Polymerase Chain Reaction (PCR) amplification of DNA can be carried out rapidly in the gas phase. In this process, hot or cold pressurized gases are delivered to a physically separate reaction chamber containing biological samples, in order to control the temperatures used for DNA denaturation, primer: template annealing, and polymerase-catalysed elongation.
It is a further object of the invention to provide for a process for subjecting biological samples to rapid thermocycling, by regulating the flow of hot or cold pressurized gas to these samples.
It is another object of the present invention to provide an apparatus suitable for carrying out gas phase amplification of DNA, using microprocessor-controlled electronic valves to regulate the flow of hot or cold pressurized gas into a thermostated biochemical reaction chamber.
It is also an object of the present invention to provide an apparatus which can subject a biological sample to rapid thermal cycling, using one or more gases at a pressure greater than one atmosphere.
It is also an object of the present invention to provide an apparatus which can subject a biological sample to rapid thermal cycling, in which pressurized air, helium, carbon dioxide, nitrogen, or argon are employed as gas phase heat transfer media.
Finally, it is an object of the present invention to provide an apparatus in which a physically separated gas heating chamber and reaction chamber are employed.
Some of the major advantages of the invention are as follows: First, the invention decreases the time needed for amplification of DNA using the Polymerase Chain Reaction by one to two orders of magnitude over any previously described process or device (see FIG. 8b). Second, the process is inherently more accurate than slower procedures, since false reaction products have practically no time to anneal and/or elongate (see FIGS. 5a, 6, 7 and 8a). Third, thermal control is as good or better than conventional heat block or hot-air thermocyclers (see FIG. 5b and 8b). Fourth, high-speed gas phase PCR is a very reliable process. Except for the electromechanical relays and valves, no moving parts are employed. Fifth, the high-speed gas phase PCR process is fully compatible with on-line detection optics, so that rapid (xcx9c1 minute) DNA amplification/detection can be carried out. The invention therefore affords rapid, accurate, and reliable DNA amplification and detection on an unprecedented time scale.
The present invention includes a high-speed process for amplifying DNA. A reaction chamber containing a biological sample, a DNA polymeras, oglionucleotide primers, and deoxynucleotide precursors is provided. The reaction chamber accepts the flow of one or more heat transfer gases. A first heat transfer gas is heated in a heating chamber which is physically separated from the reaction chamber. The heated gas is delivered to the reaction chamber at a pressure greater than the reaction chamber pressure. Heat from the heated gas is utilized to denature DNA. A second heat transfer gas, which may be the same kind as, or a different kind of gas, than the first heat transfer gas is delivered to the reaction chamber. The second heat transfer gas cools the reaction chamber to a temperature low enough to allow the denatured DNA to anneal to the oglionucleotide primers. Finally, the temperature of the reaction chamber is increased to a sufficient temperature to allow for elongation of primer:template complexes.
When hot or cold gas is delivered to a biochemical reaction chamber under the control of microprocessor-controlled electronic valves, extremely fast thermocycling is possible. By delivering, alternately, bursts of hot and cold gas to a thermostated reaction chamber, the temperature of an enzyme-catalysed reaction can be controlled. Ideally, the gas of choice is helium, but for convenience, other gases can also be employed.
The present invention can also include an apparatus for high-speed amplification of DNA. The apparatus includes a reaction chamber having a pressure, usually normal room or atmospheric pressure. A heating chamber, which is physically separated from the reaction chamber, is fluidly connected to the reaction chamber. A first container, having a heating gas at a pressure greater than the reaction chamber pressure, is fluidly connected to the heating chamber. A second container, having a cooling gas (which may be the same kind or different kind of gas as the heating gas), is fluidly connected to the reaction chamber. A cooling gas inlet valves is positioned between the second container and the reaction chamber, and a heating gas inlet valve is positioned between the first container and the reaction chamber. A programmable controller, having inputs and outputs, is used to control opening of the inlet valves. A temperature sensor is connected to an input of the controller. The controller opens and closes the inlet valves to reach or maintain a desired temperature within the reaction chamber.
If the temperature of an optimal heat transfer gas, such as helium, is raised in a chamber which is physically separated from the reaction chamber, and then delivered to the reaction chamber, then extremely fast thermocycling is possible. Even faster heat transfer is possible if cold gas is delivered to the chamber. For automated heating and cooling of the reaction chamber, preferably at least two electronic valves (hot gas valve and cold gas valve) and one or more mechanical relief valves are employed.
The design of our pressurized gas thermocycler differs in five important ways from prior art hot-air thermocyclers: (i) An optimal heat transfer gas (helium) can be employed for rapid heating/cooling of the reaction chamber. For convenience, other gases, such as air or CO2, can also be used, although heat transfer is not as fast as when helium is employed. (ii) The heat chamber and reaction chamber are physically separated in space (heat chamberxe2x89xa0reaction chamber). (iii) Microprocessor-controlled electronic valves are used to deliver pre-heated gas or pre-cooled gas to the reaction chamber. (iv) Gas is delivered to the reaction chamber at elevated pressure ( greater than 1 atm), but the biochemical reaction is sealed in a thin-walled capillary tube in which the pressure is unchanged. (v) Unlike any other thermocycler which has been preciously described, the rate-limiting step in the pressurized gas thermocycler (xcx9c1 sec/cycle) is a biochemical step (the rate of DNA Polymerase elongation) rather than the dead time of the machine itself. In fact, the pressurized gas thermocycler is so fast that, if faster thermostable DNA polymerases ( greater than 100 nucleotides/sec) were available, then 30 cycles of PCR could be carried out in less than one minute. Pressurized gas thermocyclers, but no other known devices, are capable of such high-speed DNA amplification.
In practice, it is not necessary to use pressurized helium gas for both heating and cooling the reaction chamber. Bottled pressurized carbon dioxide (CO2) gas expands and cools upon leaving its storage container. It is therefore convenient to use pressurized helium gas to heat the reaction chamber and bottled CO2 gas to cool the chamber.
For example, using hot (xcx9c180xc2x0 C.) pressurized helium gas and cold (xcx9c5xc2x0 C.) pressurized CO2 gas, which are delivered to a thermostated reaction chamber via 5V electronic valves and digital relays, it is possible to cyclically change the temperature from 92xc2x0 C. (DNA denaturation temperature) to 55xc2x0 C. (primer annealing temperature) to 72xc2x0 C. (DNA polymerase elongation temperature) in less than 2.7 seconds. With slight losses to machine dead time, 30 cycles of 92xc2x0 C./55xc2x0 C./72xc2x0 C. can be carried out in 1.3 to 2.3 minute when minimal (0 to 2 sec) D, A, and E times are used.